This year, we focused on building on and improving upon work done in the previous two years by 2017 and 2018 iGEM teams. One of our primary short term goals last year was to make our project more accessible and practical to implement in the real world by creating a liquid assay, and this assay was one of our major accomplishments this year. We also aimed to gather more quantitative data, in contrast to last year’s qualitative data in the form of plating assays, where we defined efficacy in terms of circles of E.coli. First, we used a nanodrop to determine the initial concentration of the samples we plated. To gather numerical data from our liquid assays, we utilized an absorbance assay that measured optical density, or OD. Using a spectrophotometer, we shone light through the liquid assay and a reader at the top of the machine measured the concentration of bacteria that grew under specific conditions.
Summary of Results
Future Directions
Short Term:
We hope to use the CHOP system to screen and select fluoride riboswitches with a higher affinity for fluoride, leading to more growth at relevant levels of fluoride and a greater dynamic range of detection. This would also make the system more valuable for implementation in communities where a fluoride detection system is most needed.
Long Term:
In order to bioremediate excess levels of fluoride, we envision using fluoride riboswitches to express genes that will allow for enzymes to metabolize fluoride after human consumption.
To sequester fluoride we hope to identify fluoride riboswitches with a higher affinity to fluoride and attach multiple copies of the riboswitches to nanoparticles.
For easier implementation, we hope to allow riboswitches to regulate a reporter, such as GFP, for a more obvious sign when unhealthy levels of fluoride are present in drinking water.
CHOP can be used to evaluate other transcriptional riboswitches that bind different metal ions, such as cobalt or nickel.
